1. Field of the Invention
Chagas' disease whose etiological agent is the hemoflagellate Trypanosoma cruzi is a pleomorphic clinical entity that is an important cause of morbidity and mortality in Central and South America. In some patients the infection is devastating from the very beginning with death following after a short acute phase, where meningoencephalitis and myocarditis are prominent findings. In other cases, however, the acute phase is oligosymptomatic or even inapparent and may evolve without detectable sequels. Between these two extremes, most cases course with a variable acute phase, which subsides in a few weeks, to be followed years later by digestive or cardiac symptoms or both.
The exact causes of this clinical pleomorphism are not known, although evidence from experimental infections of animals suggests that host as well as parasite factors may be involved. Concerning the etiological agent, differences have also been found among several strains of T. cruzi as to their morphology, virulence, pathogenicity, tropism and other parameters. Particularly well documented and studied are the differences between the Y and CL strains of T. cruzi. These strains, termed "polar" by Brener (1977) Pan American Scientific Organization, Scientific Publication 347:11-21, differ significantly according to various parameters which include growth in culture medium and in tissue culture, as well as in morphology and in tissue tropism.
The above mentioned differences among T. cruzi strains and recent reports support the belief that T. cruzi is not a homogeneous species. It is therefore important to be able to identify T. cruzi subgroups and relate them to observed pathology. Not only is this of interest with T. cruzi, but other organisms of the order Kinetoplastida, which share a common characteristic in the nature of their mitochondrial DNA. The mitochrondrial DNA is composed of a multiplicity of catenated, covalently closed minicircles and maxicircles organized into a two-dimensional network. This unique form of DNA offers the potential to use the electrophoretic distribution of an endonuclease digest as a diagnostic characteristic of a particular species or strain.
2. Description of the Prior Art
The minicircle component of the kDNA network of hemoflagellate protozoa consists of a few to many semihomologous sequence classes that all appear to share a constant region. Donelson et al. (1979) Plasmid 2:572-588; Steinert and Van Assel, (1980) Plasmid 3:7-17. kDNA from different hemoflagellate species have few, if any, sequences in common and kDNAs from different strains of Leishmania, or Trypanosoma vary by hybridization of cRNA and even by buoyant density analysis. Chance (1977) In: "Biochemistry of Parasites and Host Parasite Relationship." (Van den Bossche, H. ed.), Elsevier, Amsterdam, pp. 229-235; Steinert, et al. (1976) In: "The Genetic Function of Mitochondrial DNA." (Sacone and Kroon, eds.), Elsevier, Amsterdam, pp. 71-81; Newton & Burnett (1972) In: Comparative Biochemistry of Parasites. (Van den Bossche, H. ed.), Academic Press, N.Y. pp. 127-138. Classification of different strains and species of Leishmania and T. cruzi by means of kDNA buoyant analysis has been performed by Chance and Baker et al. Chance (1979) In: "Problems in the identification of parasites and their vectors." (Taylor and Muller, eds.) Blackwell Science Pub., Oxford, pp. 55-74; Baker et al., (1978) Am. J. Trop. Med. Hyg. 27:483-491. Mattei et al., (1977) FEBS Letters 74:264-268, report that different T. cruzi strains gave different kDNA restriction fingerprints in 3.5% acrylamide gels. Brack et al., (1976) In: Biochemistry of Parasites and Host-Parasite Relationships, (Van den Bossche, H., ed.), Elsevier/North Holland Biomedical Press, Amsterdam, pp. 211-218, also demonstrates the validity of species classification by restriction profiles of kDNA. Riou & Gutteridge, (1978) Biochimie 60:365-379 report the lack of qualitative differences between dDNA restriction profiles of two T. cruzi strains, while Leon et al., (1977) In: Congresso Internacional sobre Doenca de Chagas, Proceedings of an International Symposium, Fundacao Oswaldo Cruz, Rio de Janeiro, Brazil, page 77, and Leonet al., (1980) Biochim. Biophys. Acta 607:221-231, report extensive differences in kDNA restriction profiles between two isolates of the Y strain of T. cruzi and published profiles of Y strain kDNA, and conclude that minicircle digestion patterns might not be a stable and reliable criterion for strain characterization.